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Whitmire JM, Windham IH, Makobongo MO, Westland MD, Tran SC, Piñol J, Hui Y, Raheem Alkarkoushi R, Pich OQ, McGee DJ, Piazuelo MB, Melton-Celsa A, Testerman TL, Cover TL, Merrell DS. A unique Helicobacter pylori strain to study gastric cancer development. Microbiol Spectr 2025; 13:e0216324. [PMID: 39641575 PMCID: PMC11705839 DOI: 10.1128/spectrum.02163-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Accepted: 10/20/2024] [Indexed: 12/07/2024] Open
Abstract
Helicobacter pylori colonizes a majority of the human population worldwide and can trigger development of a variety of gastric diseases. Since the bacterium is classified as a carcinogen, elucidation of the characteristics of H. pylori that influence gastric carcinogenesis is a high priority. To this end, the Mongolian gerbil infection model has proven to be an important tool to study gastric cancer progression. However, only a small number of H. pylori strains have been evaluated in the gerbil model. Thus, to identify additional strains able to colonize and induce disease in this model, several H. pylori strains were used to infect Mongolian gerbils, and stomachs were harvested at multiple timepoints to assess colonization and gastric pathology. The USU101 strain reproducibly colonized Mongolian gerbils and induced gastric inflammation in the majority of the animals 1 month after infection. Adenocarcinoma or dysplasia was observed in the majority of gerbils by 2 months post-infection. To define the contribution of key virulence factors to this process, isogenic strains lacking cagA or vacA, along with restorant strains containing a wild-type (WT) copy of the genes, were studied. The ΔcagA USU101 strain colonized gerbils at levels similar to WT, but did not induce comparable levels of inflammation or disease. In contrast, the ΔvacA USU101 strain did not colonize gerbils, and the stomach pathology resembled that of the mock-infected animals. The restorant USU101 strains expressed the CagA and VacA proteins in vitro, and in vivo experiments with Mongolian gerbils showed a restoration of colonization levels and inflammation scores comparable to those observed in WT USU101. Our studies indicate that the USU101 strain is a valuable tool to study H. pylori-induced disease.IMPORTANCEGastric cancer is the fifth leading cause of cancer-related death globally; the majority of gastric cancers are associated with Helicobacter pylori infection. Infection of Mongolian gerbils with H. pylori has been shown to result in induction of gastric cancer, but few H. pylori strains have been studied in this model; this limits our ability to fully understand gastric cancer pathogenesis in humans because H. pylori strains are notoriously heterogenous. Our studies reveal that USU101 represents a unique H. pylori strain that can be added to our repertoire of strains to study gastric cancer development in the Mongolian gerbil model.
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Affiliation(s)
| | - Ian H. Windham
- Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
| | - Morris O. Makobongo
- Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
- University of South Carolina School of Medicine, Columbia, South Carolina, USA
| | | | | | - Jaume Piñol
- Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
| | - Yvonne Hui
- University of South Carolina School of Medicine, Columbia, South Carolina, USA
| | | | - Oscar Q. Pich
- Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain
- Laboratori de Recerca en Microbiologia i Malalties Infeccioses, Hospital Universitari Parc Taulí, Institut d’Investigació i Innovació Parc Taulí (I3PT-CERCA), Universitat Autònoma de Barcelona, Sabadell, Spain
| | - David J. McGee
- Department of Microbiology and Immunology, LSU Health Sciences Center-Shreveport, Shreveport, Louisiana, USA
| | | | - Angela Melton-Celsa
- Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
| | - Traci L. Testerman
- University of South Carolina School of Medicine, Columbia, South Carolina, USA
| | - Timothy L. Cover
- Vanderbilt University, Nashville, Tennessee, USA
- Vanderbilt University Medical Center, Nashville, Tennessee, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
| | - D. Scott Merrell
- Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
- School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona, USA
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Rosinke K, Starai VJ, Hoover TR. Helicobacter pylori HP0018 Has a Potential Role in the Maintenance of the Cell Envelope. Cells 2024; 13:1438. [PMID: 39273010 PMCID: PMC11394524 DOI: 10.3390/cells13171438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2024] [Revised: 08/22/2024] [Accepted: 08/23/2024] [Indexed: 09/15/2024] Open
Abstract
Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, where it can cause a variety of diseases. H. pylori uses a cluster of sheathed flagella for motility, which is required for host colonization in animal models. The flagellar sheath is continuous with the outer membrane and is found in most Helicobacter species identified to date. HP0018 is a predicted lipoprotein of unknown function that is conserved in Helicobacter species that have flagellar sheaths but is absent in Helicobacter species that have sheath-less flagella. Deletion of hp0018 in H. pylori B128 resulted in the formation of long chains of outer membrane vesicles, which were most evident in an aflagellated variant of the Δhp0018 mutant that had a frameshift mutation in fliP. Flagellated cells of the Δhp0018 mutant possessed what appeared to be a normal flagellar sheath, suggesting that HP0018 is not required for sheath formation. Cells of the Δhp0018 mutant were also less helical in shape compared to wild-type cells. A HP0018-superfolder green fluorescent fusion protein expressed in the H. pylori Δhp0018 mutant formed fluorescent foci at the cell poles and lateral sites. Co-immunoprecipitation assays with HP0018 identified two enzymes involved in the modification of the cell wall peptidoglycan, AmiA and MltD, as potential HP0018 interaction partners. HP0018 may modulate the activity of AmiA or MltD, and in the absence of HP0018, the unregulated activity of these enzymes may alter the peptidoglycan layer in a manner that results in an altered cell shape and hypervesiculation.
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Affiliation(s)
- Kyle Rosinke
- Department of Microbiology, University of Georgia, Athens, GA 30602, USA; (K.R.); (V.J.S.)
| | - Vincent J. Starai
- Department of Microbiology, University of Georgia, Athens, GA 30602, USA; (K.R.); (V.J.S.)
- Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA
| | - Timothy R. Hoover
- Department of Microbiology, University of Georgia, Athens, GA 30602, USA; (K.R.); (V.J.S.)
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3
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Sichel SR, Bratton BP, Salama NR. Distinct regions of H. pylori's bactofilin CcmA regulate protein-protein interactions to control helical cell shape. eLife 2022; 11:e80111. [PMID: 36073778 PMCID: PMC9507126 DOI: 10.7554/elife.80111] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Accepted: 09/07/2022] [Indexed: 11/26/2022] Open
Abstract
The helical shape of Helicobacter pylori cells promotes robust stomach colonization; however, how the helical shape of H. pylori cells is determined is unresolved. Previous work identified helical-cell-shape-promoting protein complexes containing a peptidoglycan-hydrolase (Csd1), a peptidoglycan precursor synthesis enzyme (MurF), a non-enzymatic homolog of Csd1 (Csd2), non-enzymatic transmembrane proteins (Csd5 and Csd7), and a bactofilin (CcmA). Bactofilins are highly conserved, spontaneously polymerizing cytoskeletal bacterial proteins. We sought to understand CcmA's function in generating the helical shape of H. pylori cells. Using CcmA deletion analysis, in vitro polymerization, and in vivo co-immunoprecipitation experiments, we identified that the bactofilin domain and N-terminal region of CcmA are required for helical cell shape and the bactofilin domain of CcmA is sufficient for polymerization and interactions with Csd5 and Csd7. We also found that CcmA's N-terminal region inhibits interaction with Csd7. Deleting the N-terminal region of CcmA increases CcmA-Csd7 interactions and destabilizes the peptidoglycan-hydrolase Csd1. Using super-resolution microscopy, we found that Csd5 recruits CcmA to the cell envelope and promotes CcmA enrichment at the major helical axis. Thus, CcmA helps organize cell-shape-determining proteins and peptidoglycan synthesis machinery to coordinate cell wall modification and synthesis, promoting the curvature required to build a helical cell.
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Affiliation(s)
- Sophie R Sichel
- Division of Human Biology, Fred Hutchinson Cancer CenterSeattleUnited States
- Molecular Medicine and Mechanisms of Disease Graduate Program, University of WashingtonSeattleUnited States
| | - Benjamin P Bratton
- Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
- Vanderbilt Institute for Infection, Immunology and InflammationNashvilleUnited States
| | - Nina R Salama
- Division of Human Biology, Fred Hutchinson Cancer CenterSeattleUnited States
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4
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Holtrup S, Greger M, Mayer B, Specht M, Waidner B. Insights Into the Helical Shape Complex of Helicobacter pylori. Front Microbiol 2022; 13:929194. [PMID: 36090072 PMCID: PMC9448923 DOI: 10.3389/fmicb.2022.929194] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/13/2022] [Indexed: 11/13/2022] Open
Abstract
One important factor that promotes the colonization of the upper digestive system of the human pathogen Helicobacter pylori is its helical cell shape. The bacteria cell shape is predominantly defined by its peptidoglycan cell wall. In rod-shaped species, PG synthesis is mediated by two dynamic molecular machines that facilitate growth along the perpendicular axis and the septum, called the elongasome and the divisome, respectively. Furthermore, many bacteria evolved additional mechanisms to locally change PG synthesis patterns to generate diverse cell shapes. Recent work characterizing cell shape mutants of Helicobacter pylori revealed a novel mechanism for the generation of a twisted helix from a rod, including PG-modifying enzymes as well as additional proteins such as the bactofilin homolog CcmA or the membrane proteins Csd5 and Csd7. In this study, we investigate the localization and dynamics of CcmA and Csd7 using live-cell imaging. We also address the question of how these change in the presence or absence of the putative interaction partners.
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Affiliation(s)
- Sven Holtrup
- LOEWE Center for Synthetic Microbiology, Marburg, Germany
- Department of Biochemistry and Chemistry, Philipps University of Marburg, Marburg, Germany
| | - Maximilian Greger
- LOEWE Center for Synthetic Microbiology, Marburg, Germany
- Department of Biochemistry and Chemistry, Philipps University of Marburg, Marburg, Germany
| | - Benjamin Mayer
- LOEWE Center for Synthetic Microbiology, Marburg, Germany
- Department of Biochemistry and Chemistry, Philipps University of Marburg, Marburg, Germany
| | - Mara Specht
- LOEWE Center for Synthetic Microbiology, Marburg, Germany
| | - Barbara Waidner
- LOEWE Center for Synthetic Microbiology, Marburg, Germany
- Department of Biochemistry and Chemistry, Philipps University of Marburg, Marburg, Germany
- *Correspondence: Barbara Waidner,
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5
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Ailloud F, Estibariz I, Suerbaum S. Evolved to vary: genome and epigenome variation in the human pathogen Helicobacter pylori. FEMS Microbiol Rev 2021; 45:5900976. [PMID: 32880636 DOI: 10.1093/femsre/fuaa042] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 08/31/2020] [Indexed: 12/24/2022] Open
Abstract
Helicobacter pylori is a Gram-negative, spiral shaped bacterium that selectively and chronically infects the gastric mucosa of humans. The clinical course of this infection can range from lifelong asymptomatic infection to severe disease, including peptic ulcers or gastric cancer. The high mutation rate and natural competence typical of this species are responsible for massive inter-strain genetic variation exceeding that observed in all other bacterial human pathogens. The adaptive value of such a plastic genome is thought to derive from a rapid exploration of the fitness landscape resulting in fast adaptation to the changing conditions of the gastric environment. Nevertheless, diversity is also lost through recurrent bottlenecks and H. pylori's lifestyle is thus a perpetual race to maintain an appropriate pool of standing genetic variation able to withstand selection events. Another aspect of H. pylori's diversity is a large and variable repertoire of restriction-modification systems. While not yet completely understood, methylome evolution could generate enough transcriptomic variation to provide another intricate layer of adaptive potential. This review provides an up to date synopsis of this rapidly emerging area of H. pylori research that has been enabled by the ever-increasing throughput of Omics technologies and a multitude of other technological advances.
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Affiliation(s)
- Florent Ailloud
- Max von Pettenkofer Institute, Faculty of Medicine, LMU München, Pettenkoferstr. 9a, 80336 München, Germany
| | - Iratxe Estibariz
- Max von Pettenkofer Institute, Faculty of Medicine, LMU München, Pettenkoferstr. 9a, 80336 München, Germany
| | - Sebastian Suerbaum
- Max von Pettenkofer Institute, Faculty of Medicine, LMU München, Pettenkoferstr. 9a, 80336 München, Germany.,DZIF Deutsches Zentrum für Infektionsforschung, Partner Site Munich, Pettenkoferstr. 9a, 80336 München, Germany.,National Reference Center for Helicobacter pylori, Pettenkoferstr. 9a, 80336 München, Germany
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6
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Taylor JA, Bratton BP, Sichel SR, Blair KM, Jacobs HM, DeMeester KE, Kuru E, Gray J, Biboy J, VanNieuwenhze MS, Vollmer W, Grimes CL, Shaevitz JW, Salama NR. Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori. eLife 2020; 9:52482. [PMID: 31916938 PMCID: PMC7012605 DOI: 10.7554/elife.52482] [Citation(s) in RCA: 39] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2019] [Accepted: 01/07/2020] [Indexed: 12/15/2022] Open
Abstract
Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape. Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria’s cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.
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Affiliation(s)
- Jennifer A Taylor
- Department of Microbiology, University of Washington, Seattle, United States.,Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, United States
| | - Benjamin P Bratton
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, United States.,Department of Molecular Biology, Princeton University, Princeton, United States
| | - Sophie R Sichel
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular Medicine and Mechanisms of Disease Graduate Program, University of Washington, Seattle, United States
| | - Kris M Blair
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, United States
| | - Holly M Jacobs
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, United States
| | - Kristen E DeMeester
- Department of Chemistry and Biochemistry, University of Delaware, Newark, United States
| | - Erkin Kuru
- Department of Genetics, Harvard Medical School, Boston, United States
| | - Joe Gray
- Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Jacob Biboy
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
| | | | - Waldemar Vollmer
- Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Catherine L Grimes
- Department of Chemistry and Biochemistry, University of Delaware, Newark, United States.,Department of Biological Sciences, University of Delaware, Newark, United States
| | - Joshua W Shaevitz
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, United States.,Department of Physics, Princeton University, Princeton, United States
| | - Nina R Salama
- Department of Microbiology, University of Washington, Seattle, United States.,Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, United States.,Molecular Medicine and Mechanisms of Disease Graduate Program, University of Washington, Seattle, United States.,Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, United States
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7
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Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori. Nat Commun 2019; 10:5357. [PMID: 31767852 PMCID: PMC6877725 DOI: 10.1038/s41467-019-13352-6] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Accepted: 10/31/2019] [Indexed: 02/08/2023] Open
Abstract
Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC. Some bacteria can take up DNA molecules from the environment. Here, Damke et al. identify a DNA-binding protein in Helicobacter pylori that is required for DNA import into the periplasm and that interacts with an inner-membrane channel that translocates the DNA into the cytoplasm.
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8
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A Genome-Wide Helicobacter pylori Morphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex. J Bacteriol 2019; 201:JB.00724-18. [PMID: 31036730 DOI: 10.1128/jb.00724-18] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Accepted: 04/26/2019] [Indexed: 12/16/2022] Open
Abstract
Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology.IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.
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9
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Stingl K, Koraimann G. Prokaryotic Information Games: How and When to Take up and Secrete DNA. Curr Top Microbiol Immunol 2019. [PMID: 29536355 DOI: 10.1007/978-3-319-75241-9_3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Besides transduction via bacteriophages natural transformation and bacterial conjugation are the most important mechanisms driving bacterial evolution and horizontal gene spread. Conjugation systems have evolved in eubacteria and archaea. In Gram-positive and Gram-negative bacteria, cell-to-cell DNA transport is typically facilitated by a type IV secretion system (T4SS). T4SSs also mediate uptake of free DNA in Helicobacter pylori, while most transformable bacteria use a type II secretion/type IV pilus system. In this chapter, we focus on how and when bacteria "decide" that such a DNA transport apparatus is to be expressed and assembled in a cell that becomes competent. Development of DNA uptake competence and DNA transfer competence is driven by a variety of stimuli and often involves intricate regulatory networks leading to dramatic changes in gene expression patterns and bacterial physiology. In both cases, genetically homogeneous populations generate a distinct subpopulation that is competent for DNA uptake or DNA transfer or might uniformly switch into competent state. Phenotypic conversion from one state to the other can rely on bistable genetic networks that are activated stochastically with the integration of external signaling molecules. In addition, we discuss principles of DNA uptake processes in naturally transformable bacteria and intend to understand the exceptional use of a T4SS for DNA import in the gastric pathogen H. pylori. Realizing the events that trigger developmental transformation into competence within a bacterial population will eventually help to create novel and effective therapies against the transmission of antibiotic resistances among pathogens.
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Affiliation(s)
- Kerstin Stingl
- National Reference Laboratory for Campylobacter, Department Biological Safety, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, 12277, Berlin, Germany.
| | - Günther Koraimann
- Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010, Graz, Austria.
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10
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Grohmann E, Christie PJ, Waksman G, Backert S. Type IV secretion in Gram-negative and Gram-positive bacteria. Mol Microbiol 2018; 107:455-471. [PMID: 29235173 PMCID: PMC5796862 DOI: 10.1111/mmi.13896] [Citation(s) in RCA: 237] [Impact Index Per Article: 33.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2017] [Revised: 12/07/2017] [Accepted: 12/09/2017] [Indexed: 02/06/2023]
Abstract
Type IV secretion systems (T4SSs) are versatile multiprotein nanomachines spanning the entire cell envelope in Gram-negative and Gram-positive bacteria. They play important roles through the contact-dependent secretion of effector molecules into eukaryotic hosts and conjugative transfer of mobile DNA elements as well as contact-independent exchange of DNA with the extracellular milieu. In the last few years, many details on the molecular mechanisms of T4SSs have been elucidated. Exciting structures of T4SS complexes from Escherichia coli plasmids R388 and pKM101, Helicobacter pylori and Legionella pneumophila have been solved. The structure of the F-pilus was also reported and surprisingly revealed a filament composed of pilin subunits in 1:1 stoichiometry with phospholipid molecules. Many new T4SSs have been identified and characterized, underscoring the structural and functional diversity of this secretion superfamily. Complex regulatory circuits also have been shown to control T4SS machine production in response to host cell physiological status or a quorum of bacterial recipient cells in the vicinity. Here, we summarize recent advances in our knowledge of 'paradigmatic' and emerging systems, and further explore how new basic insights are aiding in the design of strategies aimed at suppressing T4SS functions in bacterial infections and spread of antimicrobial resistances.
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Affiliation(s)
- Elisabeth Grohmann
- Beuth University of Applied Sciences Berlin, Life Sciences and Technology, D-13347 Berlin, Germany
| | - Peter J. Christie
- Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, 6431 Fannin St, Houston, Texas 77030, USA
| | - Gabriel Waksman
- Institute of Structural and Molecular Biology, University College London and Birkbeck College, London WC1E 7HX, United Kingdom
| | - Steffen Backert
- Friedrich Alexander University Erlangen-Nuremberg, Department of Biology, Division of Microbiology, Staudtstrasse 5, D-91058 Erlangen, Germany
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11
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Gall A, Gaudet RG, Gray-Owen SD, Salama NR. TIFA Signaling in Gastric Epithelial Cells Initiates the cag Type 4 Secretion System-Dependent Innate Immune Response to Helicobacter pylori Infection. mBio 2017; 8:e01168-17. [PMID: 28811347 PMCID: PMC5559637 DOI: 10.1128/mbio.01168-17] [Citation(s) in RCA: 97] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Accepted: 07/10/2017] [Indexed: 12/14/2022] Open
Abstract
Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, causing inflammation which, in some cases, leads to gastric ulcers and cancer. The clinical outcome of infection depends on a complex interplay of bacterial, host genetic, and environmental factors. Although H. pylori is recognized by both the innate and adaptive immune systems, this rarely results in bacterial clearance. Gastric epithelial cells are the first line of defense against H. pylori and alert the immune system to bacterial presence. Cytosolic delivery of proinflammatory bacterial factors through the cag type 4 secretion system (cag-T4SS) has long been appreciated as the major mechanism by which gastric epithelial cells detect H. pylori Classically attributed to the peptidoglycan sensor NOD1, recent work has highlighted the role of NOD1-independent pathways in detecting H. pylori; however, the bacterial and host factors involved have remained unknown. Here, we show that bacterially derived heptose-1,7-bisphosphate (HBP), a metabolic precursor in lipopolysaccharide (LPS) biosynthesis, is delivered to the host cytosol through the cag-T4SS, where it activates the host tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA)-dependent cytosolic surveillance pathway. This response, which is independent of NOD1, drives robust NF-κB-dependent inflammation within hours of infection and precedes NOD1 activation. We also found that the CagA toxin contributes to the NF-κB-driven response subsequent to TIFA and NOD1 activation. Taken together, our results indicate that the sequential activation of TIFA, NOD1, and CagA delivery drives the initial inflammatory response in gastric epithelial cells, orchestrating the subsequent recruitment of immune cells and leading to chronic gastritis.IMPORTANCEH. pylori is a globally prevalent cause of gastric and duodenal ulcers and cancer. H. pylori antibiotic resistance is rapidly increasing, and a vaccine remains elusive. The earliest immune response to H. pylori is initiated by gastric epithelial cells and sets the stage for the subsequent immunopathogenesis. This study revealed that host TIFA and H. pylori-derived HBP are critical effectors of innate immune signaling that account for much of the inflammatory response to H. pylori in gastric epithelial cells. HBP is delivered to the host cell via the cag-T4SS at a time point that precedes activation of the previously described NOD1 and CagA inflammatory pathways. Manipulation of the TIFA-driven immune response in the host and/or targeting of ADP-heptose biosynthesis enzymes in H. pylori may therefore provide novel strategies that may be therapeutically harnessed to achieve bacterial clearance.
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Affiliation(s)
- Alevtina Gall
- Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, Washington, USA
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
| | - Ryan G Gaudet
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- Howard Hughes Medical Institute and Departments of Microbial Pathogenesis and of Immunobiology, Yale University, New Haven, Connecticut, USA
| | - Scott D Gray-Owen
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Nina R Salama
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
- Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, USA
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Hutton ML, D'Costa K, Rossiter AE, Wang L, Turner L, Steer DL, Masters SL, Croker BA, Kaparakis-Liaskos M, Ferrero RL. A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization. Front Cell Infect Microbiol 2017; 7:219. [PMID: 28634572 PMCID: PMC5460342 DOI: 10.3389/fcimb.2017.00219] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2017] [Accepted: 05/11/2017] [Indexed: 12/12/2022] Open
Abstract
The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.
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Affiliation(s)
- Melanie L. Hutton
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - Kimberley D'Costa
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - Amanda E. Rossiter
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - Lin Wang
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - Lorinda Turner
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - David L. Steer
- Monash Biomedical Proteomics Facility, Monash UniversityMelbourne, VIC, Australia
| | - Seth L. Masters
- Inflammation Division, The Walter and Eliza Hall InstituteMelbourne, VIC, Australia
| | - Ben A. Croker
- Inflammation Division, The Walter and Eliza Hall InstituteMelbourne, VIC, Australia
| | - Maria Kaparakis-Liaskos
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
| | - Richard L. Ferrero
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical ResearchMelbourne, VIC, Australia
- Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash UniversityMelbourne, VIC, Australia
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Zawilak-Pawlik A, Zakrzewska-Czerwińska J. Recent Advances in Helicobacter pylori Replication: Possible Implications in Adaptation to a Pathogenic Lifestyle and Perspectives for Drug Design. Curr Top Microbiol Immunol 2017; 400:73-103. [PMID: 28124150 DOI: 10.1007/978-3-319-50520-6_4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
DNA replication is an important step in the life cycle of every cell that ensures the continuous flow of genetic information from one generation to the next. In all organisms, chromosome replication must be coordinated with overall cell growth. Helicobacter pylori growth strongly depends on its interaction with the host, particularly with the gastric epithelium. Moreover, H. pylori actively searches for an optimal microniche within a stomach, and it has been shown that not every microniche equally supports growth of this bacterium. We postulate that besides nutrients, H. pylori senses different, unknown signals, which presumably also affect chromosome replication to maintain H. pylori propagation at optimal ratio allowing H. pylori to establish a chronic, lifelong infection. Thus, H. pylori chromosome replication and particularly the regulation of this process might be considered important for bacterial pathogenesis. Here, we summarize our current knowledge of chromosome and plasmid replication in H. pylori and discuss the mechanisms responsible for regulating this key cellular process. The results of extensive studies conducted thus far allow us to propose common and unique traits in H. pylori chromosome replication. Interestingly, the repertoire of proteins involved in replication in H. pylori is significantly different to that in E. coli, strongly suggesting that novel factors are engaged in H. pylori chromosome replication and could represent attractive drug targets.
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Affiliation(s)
- Anna Zawilak-Pawlik
- Department of Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Ul. Weigla 12, 53-114, Wrocław, Poland.
| | - Jolanta Zakrzewska-Czerwińska
- Department of Microbiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Ul. Weigla 12, 53-114, Wrocław, Poland
- Department of Molecular Microbiology, Faculty of Biotechnology, University of Wrocław, Ul. Joliot-Curie 14A, 50-383, Wrocław, Poland
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Turner L, Praszkier J, Hutton ML, Steer D, Ramm G, Kaparakis-Liaskos M, Ferrero RL. Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant. Helicobacter 2015; 20:269-83. [PMID: 25669590 DOI: 10.1111/hel.12196] [Citation(s) in RCA: 76] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
BACKGROUND Multiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. METHODS H. pylori ΔtolB, Δpal and ΔtolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. RESULTS H. pylori ΔtolB and Δpal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H. pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H. pylori ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. CONCLUSIONS This work demonstrates that TolB and Pal are important for membrane integrity in H. pylori. Moreover, it shows how H. pylori tolB-pal genes may be manipulated to develop "hypervesiculating" strains for vaccine purposes.
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Affiliation(s)
- Lorinda Turner
- Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia
| | - Judyta Praszkier
- Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia
| | - Melanie L Hutton
- Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia
| | - David Steer
- Monash Biomedical Proteomics Facility, Monash University, Wellington Road, Clayton, (3800), Victoria, Australia
| | - Georg Ramm
- Monash Micro Imaging, Monash University, Clayton, Victoria, Australia
| | - Maria Kaparakis-Liaskos
- Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia
| | - Richard L Ferrero
- Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia
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Abstract
The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal. Bacterial biofilms are ubiquitous in nature, but the mechanisms governing their assembly and spatial organization are not fully understood. Bacterial communication through quorum sensing has been shown to influence biofilm growth through the regulation of biofilm genes. Our study revealed a new role for quorum sensing in biofilms through rapid chemotactic responses to quorum signals. Specifically, we studied how chemorepulsion of Helicobacter pylori from the universal quorum signal autoinducer-2 (AI-2) shapes the spatial organization of its biofilms. We demonstrate that the chemorepulsive response of H. pylori to AI-2 is necessary to promote its dispersal from biofilms grown on both abiotic and biotic surfaces and is sufficient to promote dispersal in a chemotaxis-dependent manner. This work has broad implications for understanding the mechanisms by which endogenously produced microbial compounds shape the assembly and spatial organization of microbial communities in their environments.
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Fernandez-Gonzalez E, Backert S. DNA transfer in the gastric pathogen Helicobacter pylori. J Gastroenterol 2014; 49:594-604. [PMID: 24515309 DOI: 10.1007/s00535-014-0938-y] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Accepted: 01/16/2014] [Indexed: 02/08/2023]
Abstract
The gastric pathogen Helicobacter pylori is one of the most genetically diverse bacteria. Recombination and DNA transfer contribute to its genetic variability and enhance host adaptation. Among the strategies described to increase genetic diversity in bacteria, DNA transfer by conjugation is one of the best characterized. Using this mechanism, a fragment of DNA from a donor cell can be transferred to a recipient, always mediated by a conjugative nucleoprotein complex, which is evolutionarily related to type IV secretion systems (T4SSs). Interestingly, the H. pylori chromosomes can encode up to four T4SSs, including the cagPAI, comB, tfs3, and tfs4 genes, some of which are known to promote chronic H. pylori infection. The T4SS encoded by the cagPAI mediates the injection of the effector protein CagA and proinflammatory signaling, and the comB system is involved in DNA uptake from the environment. However, the role of tfs3 and tfs4 is not yet clear. The presence of a functional XerD tyrosine recombinase and 5'AAAGAATG-3' border sequences as well as two putative conjugative relaxases (Rlx1 and Rlx2), a coupling protein (TraG), and a chromosomal region carrying a putative origin of transfer (oriT) suggest the existence of a DNA transfer apparatus in tfs4. Moreover, a conjugation-like DNA transfer mechanism in H. pylori has already been described in vitro, but whether this occurs in vivo is still unknown. Some extrachromosomal plasmids and phages are also present in various H. pylori strains. Genetic exchange among plasmids and chromosomes, and involved DNA mobilization events, could explain part of H. pylori's genetic diversity. Here, we review our knowledge about the possible DNA transfer mechanisms in H. pylori and its implications in bacterial adaptation to the host environment.
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Affiliation(s)
- Esther Fernandez-Gonzalez
- Division of Microbiology, Department of Biology, Friedrich Alexander University Erlangen/Nuremberg, Staudtstr. 5, 91058, Erlangen, Germany
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17
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Rubin EJ, O'Brien JP, Ivanov PL, Brodbelt JS, Trent MS. Identification of a broad family of lipid A late acyltransferases with non-canonical substrate specificity. Mol Microbiol 2014; 91:887-99. [PMID: 24372821 DOI: 10.1111/mmi.12501] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/19/2013] [Indexed: 12/21/2022]
Abstract
Most Gram-negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine-step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyses addition of a C12:0 or C14:0 acyl chain to the 3'-linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologues in Campylobacter jejuni and Wolinella succinogenes can act before the 2' secondary acyltransferase, LpxL, as well as the 3-deoxy-d-manno-octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homologue, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homologue.
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Affiliation(s)
- Erica J Rubin
- Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA
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Wang W, Ding J, Zhang Y, Hu Y, Wang DC. Structural insights into the unique single-stranded DNA-binding mode of Helicobacter pylori DprA. Nucleic Acids Res 2013; 42:3478-91. [PMID: 24369431 PMCID: PMC3950713 DOI: 10.1093/nar/gkt1334] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to ‘open’ the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA–ssDNA.
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Affiliation(s)
- Wei Wang
- The National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China
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Phylogeographic origin of Helicobacter pylori determines host-adaptive responses upon coculture with gastric epithelial cells. Infect Immun 2013; 81:2468-77. [PMID: 23630959 DOI: 10.1128/iai.01182-12] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
While Helicobacter pylori infects over 50% of the world's population, the mechanisms involved in the development of gastric disease are not fully understood. Bacterial, host, and environmental factors play a role in disease outcome. To investigate the role of bacterial factors in H. pylori pathogenesis, global gene expression of six H. pylori isolates was analyzed during coculture with gastric epithelial cells. Clustering analysis of six Colombian clinical isolates from a region with low gastric cancer risk and a region with high gastric cancer risk segregated strains based on their phylogeographic origin. One hundred forty-six genes had increased expression in European strains, while 350 genes had increased expression in African strains. Differential expression was observed in genes associated with motility, pathogenicity, and other adaptations to the host environment. European strains had greater expression of the virulence factors cagA, vacA, and babB and were associated with increased gastric histologic lesions in patients. In AGS cells, European strains promoted significantly higher interleukin-8 (IL-8) expression than did African strains. African strains significantly induced apoptosis, whereas only one European strain significantly induced apoptosis. Our data suggest that gene expression profiles of clinical isolates can discriminate strains by phylogeographic origin and that these profiles are associated with changes in expression of the proinflammatory and protumorigenic cytokine IL-8 and levels of apoptosis in host epithelial cells. These findings support the hypothesis that bacterial factors determined by the phylogeographic origin of H. pylori strains may promote increased gastric disease.
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Alka K, Windle HJ, Cornally D, Ryan BJ, Henehan GTM. A short chain NAD(H)-dependent alcohol dehydrogenase (HpSCADH) from Helicobacter pylori: a role in growth under neutral and acidic conditions. Int J Biochem Cell Biol 2013; 45:1347-55. [PMID: 23583739 DOI: 10.1016/j.biocel.2013.04.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2012] [Revised: 03/27/2013] [Accepted: 04/03/2013] [Indexed: 11/18/2022]
Abstract
Toxic aldehydes produced by alcohol dehydrogenases have been implicated in the pathogenesis of Helicobacter pylori-related damage to the gastric mucosa. Despite this, the enzymes that might be responsible for producing such aldehydes have not been fully described. It was, therefore, of considerable interest to characterize the alcohol oxidizing enzymes in this pathogen. Previous work in this laboratory characterized two such H. pylori enzymes that had broad specificity for a range of aromatic alcohol substrates. However, an enzyme with specificity for aliphatic alcohols is likely to be required in order that H. pylori can metabolize the wide range of substrates encountered in the gastric mucosa. In this study we describe HpSCADH, an alcohol dehydrogenase from H. pylori 26695 with broad specificity for aliphatic alcohols. HpSCADH was classified in the cD1e subfamily of classical short chain alcohol dehydrogenases. The enzyme was a monomer of approximately 29kDa with a preference for NAD(+) as cofactor. Pyrazole was found to be a competitive inhibitor of HpSCADH. The physiological role of this enzyme was explored by construction of an HpSCADH isogenic mutant. At pH 7.0 the mutant showed reduced growth which became more pronounced when the pH was lowered to 5.0. When pyrazole was added to wild type H. pylori cells it caused growth profiles to be reduced to match those of the isogenic mutant suggesting that HpSCADH inhibition alone was responsible for growth impairment. Taken together, the data relating to the alcohol metabolizing enzymes of this pathogen indicate that they play an important role in H. pylori growth and adaptation to acidic environments. The therapeutic potential of targeting H. pylori alcohol dehydrogenases is discussed.
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Affiliation(s)
- Kumari Alka
- School of Food Science and Environmental Health, Dublin Institute of Technology, Marlborough Street, Dublin 1, Ireland
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Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases. J Bacteriol 2012; 194:3407-16. [PMID: 22522893 DOI: 10.1128/jb.00113-12] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.
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Cullen TW, Giles DK, Wolf LN, Ecobichon C, Boneca IG, Trent MS. Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa. PLoS Pathog 2011; 7:e1002454. [PMID: 22216004 PMCID: PMC3245313 DOI: 10.1371/journal.ppat.1002454] [Citation(s) in RCA: 159] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2011] [Accepted: 11/08/2011] [Indexed: 12/11/2022] Open
Abstract
Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4'-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4'-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4'-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4'-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.
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Affiliation(s)
- Thomas W. Cullen
- Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, United States of America
| | - David K. Giles
- Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, United States of America
| | - Lindsey N. Wolf
- Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, United States of America
| | - Chantal Ecobichon
- Institut Pasteur, Group Biology and Genetics of the Bacterial Cell Wall, Paris, France
- INSERM, Groupe Avenir, Paris, France
| | - Ivo G. Boneca
- Institut Pasteur, Group Biology and Genetics of the Bacterial Cell Wall, Paris, France
- INSERM, Groupe Avenir, Paris, France
| | - M. Stephen Trent
- Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas, United States of America
- The Institute of Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, United States of America
- * E-mail:
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Kawai M, Furuta Y, Yahara K, Tsuru T, Oshima K, Handa N, Takahashi N, Yoshida M, Azuma T, Hattori M, Uchiyama I, Kobayashi I. Evolution in an oncogenic bacterial species with extreme genome plasticity: Helicobacter pylori East Asian genomes. BMC Microbiol 2011; 11:104. [PMID: 21575176 PMCID: PMC3120642 DOI: 10.1186/1471-2180-11-104] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2010] [Accepted: 05/16/2011] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND The genome of Helicobacter pylori, an oncogenic bacterium in the human stomach, rapidly evolves and shows wide geographical divergence. The high incidence of stomach cancer in East Asia might be related to bacterial genotype. We used newly developed comparative methods to follow the evolution of East Asian H. pylori genomes using 20 complete genome sequences from Japanese, Korean, Amerind, European, and West African strains. RESULTS A phylogenetic tree of concatenated well-defined core genes supported divergence of the East Asian lineage (hspEAsia; Japanese and Korean) from the European lineage ancestor, and then from the Amerind lineage ancestor. Phylogenetic profiling revealed a large difference in the repertoire of outer membrane proteins (including oipA, hopMN, babABC, sabAB and vacA-2) through gene loss, gain, and mutation. All known functions associated with molybdenum, a rare element essential to nearly all organisms that catalyzes two-electron-transfer oxidation-reduction reactions, appeared to be inactivated. Two pathways linking acetyl~CoA and acetate appeared intact in some Japanese strains. Phylogenetic analysis revealed greater divergence between the East Asian (hspEAsia) and the European (hpEurope) genomes in proteins in host interaction, specifically virulence factors (tipα), outer membrane proteins, and lipopolysaccharide synthesis (human Lewis antigen mimicry) enzymes. Divergence was also seen in proteins in electron transfer and translation fidelity (miaA, tilS), a DNA recombinase/exonuclease that recognizes genome identity (addA), and DNA/RNA hybrid nucleases (rnhAB). Positively selected amino acid changes between hspEAsia and hpEurope were mapped to products of cagA, vacA, homC (outer membrane protein), sotB (sugar transport), and a translation fidelity factor (miaA). Large divergence was seen in genes related to antibiotics: frxA (metronidazole resistance), def (peptide deformylase, drug target), and ftsA (actin-like, drug target). CONCLUSIONS These results demonstrate dramatic genome evolution within a species, especially in likely host interaction genes. The East Asian strains appear to differ greatly from the European strains in electron transfer and redox reactions. These findings also suggest a model of adaptive evolution through proteome diversification and selection through modulation of translational fidelity. The results define H. pylori East Asian lineages and provide essential information for understanding their pathogenesis and designing drugs and therapies that target them.
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Affiliation(s)
- Mikihiko Kawai
- Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan
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Gilbreath JJ, Cody WL, Merrell DS, Hendrixson DR. Change is good: variations in common biological mechanisms in the epsilonproteobacterial genera Campylobacter and Helicobacter. Microbiol Mol Biol Rev 2011; 75:84-132. [PMID: 21372321 PMCID: PMC3063351 DOI: 10.1128/mmbr.00035-10] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Microbial evolution and subsequent species diversification enable bacterial organisms to perform common biological processes by a variety of means. The epsilonproteobacteria are a diverse class of prokaryotes that thrive in diverse habitats. Many of these environmental niches are labeled as extreme, whereas other niches include various sites within human, animal, and insect hosts. Some epsilonproteobacteria, such as Campylobacter jejuni and Helicobacter pylori, are common pathogens of humans that inhabit specific regions of the gastrointestinal tract. As such, the biological processes of pathogenic Campylobacter and Helicobacter spp. are often modeled after those of common enteric pathogens such as Salmonella spp. and Escherichia coli. While many exquisite biological mechanisms involving biochemical processes, genetic regulatory pathways, and pathogenesis of disease have been elucidated from studies of Salmonella spp. and E. coli, these paradigms often do not apply to the same processes in the epsilonproteobacteria. Instead, these bacteria often display extensive variation in common biological mechanisms relative to those of other prototypical bacteria. In this review, five biological processes of commonly studied model bacterial species are compared to those of the epsilonproteobacteria C. jejuni and H. pylori. Distinct differences in the processes of flagellar biosynthesis, DNA uptake and recombination, iron homeostasis, interaction with epithelial cells, and protein glycosylation are highlighted. Collectively, these studies support a broader view of the vast repertoire of biological mechanisms employed by bacteria and suggest that future studies of the epsilonproteobacteria will continue to provide novel and interesting information regarding prokaryotic cellular biology.
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Affiliation(s)
- Jeremy J. Gilbreath
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
| | - William L. Cody
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
| | - D. Scott Merrell
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
| | - David R. Hendrixson
- Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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25
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Vitamin B6 is required for full motility and virulence in Helicobacter pylori. mBio 2010; 1. [PMID: 21151756 PMCID: PMC3000542 DOI: 10.1128/mbio.00112-10] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2010] [Accepted: 07/15/2010] [Indexed: 12/16/2022] Open
Abstract
Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori, we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B6 biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B6 biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B6 is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. Thus, for the first time, we identify vitamin B6 biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens. Approximately half of the world’s population is infected with H. pylori, yet how H. pylori bacteria establish chronic infections in human hosts remains elusive. From gene array studies, we identified two genes as representing potentially novel colonization factors for H. pylori. These genes encoded enzymes involved in the synthesis of vitamin B6, an important molecule for many metabolic reactions in living organisms. Little is currently known regarding vitamin B6 biosynthesis in human pathogens. We showed that mutant H. pylori bacteria lacking an enzyme involved in de novo vitamin B6 biosynthesis, PdxA, were unable to synthesize motility appendages (flagella) and were unable to establish chronic colonization in mice. Thus, this work identifies vitamin B6 biosynthesis enzymes as novel virulence factors for bacterial pathogens. Interestingly, a number of human pathogens, but not their mammalian hosts, possess these genes, which suggests that Pdx enzymes may represent ideal candidates for new therapeutic targets.
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Sycuro LK, Pincus Z, Gutierrez KD, Biboy J, Stern CA, Vollmer W, Salama NR. Peptidoglycan crosslinking relaxation promotes Helicobacter pylori's helical shape and stomach colonization. Cell 2010; 141:822-33. [PMID: 20510929 PMCID: PMC2920535 DOI: 10.1016/j.cell.2010.03.046] [Citation(s) in RCA: 205] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2009] [Revised: 01/29/2010] [Accepted: 03/19/2010] [Indexed: 02/07/2023]
Abstract
The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.
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Affiliation(s)
- Laura K. Sycuro
- Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA 98195 USA
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA
| | - Zachary Pincus
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520 USA
| | | | - Jacob Biboy
- Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH UK
| | - Chelsea A. Stern
- Department of Microbiology, University of Washington, Seattle, WA 98195 USA
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA
| | - Waldemar Vollmer
- Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, NE2 4HH UK
| | - Nina R. Salama
- Department of Microbiology, University of Washington, Seattle, WA 98195 USA
- Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 USA
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27
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Analysis of protein expression regulated by the Helicobacter pylori ArsRS two-component signal transduction system. J Bacteriol 2010; 192:2034-43. [PMID: 20154125 DOI: 10.1128/jb.01703-08] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Previous studies have shown that the Helicobacter pylori ArsRS two-component signal transduction system contributes to acid-responsive gene expression. To identify additional members of the ArsRS regulon and further investigate the regulatory role of the ArsRS system, we analyzed protein expression in wild-type and arsS null mutant strains. Numerous proteins were differentially expressed in an arsS mutant strain compared to a wild-type strain when the bacteria were cultured at pH 5.0 and also when they were cultured at pH 7.0. Genes encoding 14 of these proteins were directly regulated by the ArsRS system, based on observed binding of ArsR to the relevant promoter regions. The ArsRS-regulated proteins identified in this study contribute to acid resistance (urease and amidase), acetone metabolism (acetone carboxylase), resistance to oxidative stress (thioredoxin reductase), quorum sensing (Pfs), and several other functions. These results provide further definition of the ArsRS regulon and underscore the importance of the ArsRS system in regulating expression of H. pylori proteins during bacterial growth at both neutral pH and acidic pH.
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Gaskin DJH, van Vliet AHM. Random mutagenesis strategies for Campylobacter and Helicobacter species. Methods Mol Biol 2010; 634:37-52. [PMID: 20676974 DOI: 10.1007/978-1-60761-652-8_3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/29/2023]
Abstract
Campylobacter and Helicobacter species are important pathogens in man and animals. The study of their virulence and physiology has been difficult due to the lack of tractable genetic tools, since many of the techniques established in Escherichia coli and related species were found to be non-functional in Campylobacter and Helicobacter species. The advent of functional genomics techniques in the last decade has been accompanied by the development of genetic tools, which take advantage of specific features of Campylobacter and Helicobacter, like natural transformation. This has allowed for the construction of random mutant libraries based on in vitro transposition or ligated loops followed by natural transformation and recombination, thus circumventing selection against sequences when cloning or passaging libraries through E. coli. Uses of the techniques have been in the study of motility, gene expression, and gene essentiality. In this chapter, we discuss the approaches and techniques used for the construction of random mutant libraries in both Campylobacter and Helicobacter.
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Cag3 is a novel essential component of the Helicobacter pylori Cag type IV secretion system outer membrane subcomplex. J Bacteriol 2009; 191:7343-52. [PMID: 19801411 DOI: 10.1128/jb.00946-09] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein.
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30
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Amundsen SK, Fero J, Salama NR, Smith GR. Dual nuclease and helicase activities of Helicobacter pylori AddAB are required for DNA repair, recombination, and mouse infectivity. J Biol Chem 2009; 284:16759-16766. [PMID: 19395381 PMCID: PMC2719311 DOI: 10.1074/jbc.m109.005587] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2009] [Revised: 04/09/2009] [Indexed: 01/18/2023] Open
Abstract
Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts of Escherichia coli expressing H. pylori addA(NUC)B or addAB(NUC) mutants unwound DNA but had approximately half of the exonuclease activity of wild-type AddAB; the addA(NUC)B(NUC) double mutant lacked detectable nuclease activity but retained helicase activity. Extracts with AddA(HEL)B lacked detectable helicase and nuclease activity. H. pylori with the single nuclease domain mutations were somewhat less sensitive to the DNA-damaging agent ciprofloxacin than the corresponding deletion mutant, suggesting that residual nuclease activity promotes limited DNA repair. The addA(NUC) and addA(HEL) mutants colonized the stomach less efficiently than the wild type; addB(NUC) showed partial attenuation. E. coli DeltarecBCD expressing H. pylori addAB was recombination-deficient unless H. pylori recA was also expressed, suggesting a species-specific interaction between AddAB and RecA and also that H. pylori AddAB participates in both DNA repair and recombination. These results support a role for both the AddAB nuclease and helicase in DNA repair and promoting infectivity.
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Affiliation(s)
| | - Jutta Fero
- Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
| | - Nina R Salama
- Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
| | - Gerald R Smith
- From the Divisions of Basic Sciences, Seattle, Washington 98109.
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31
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Recombination-based in vivo expression technology identifies Helicobacter pylori genes important for host colonization. Infect Immun 2008; 76:5632-44. [PMID: 18794279 DOI: 10.1128/iai.00627-08] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Here we undertook to identify colonization and gastric disease-promoting factors of the human gastric pathogen Helicobacter pylori as genes that were induced in response to the stomach environment. Using recombination-based in vivo expression technology (RIVET), we identified six promoters induced in the host compared to laboratory conditions. Three of these promoters, designated Pivi10, Pivi66, and Pivi77, regulate genes that H. pylori may use to interact with other microbes or the host. Pivi10 likely regulates the mobA, mobB, and mobD genes, which have potential roles in horizontal gene transfer through plasmid mobilization. Pivi66 occurs in the cytotoxin-associated gene pathogenicity island, a genomic region known to be associated with more severe disease outcomes, and likely regulates cagZ, virB11, and virD4. Pivi77 likely regulates HP0289, an uncharacterized paralogue of the vacA cytotoxin gene. We assessed the roles of a subset of these genes in colonization by creating deletion mutants and analyzing them in single-strain and coinfection experiments. We found that a mobABD mutant was defective for murine host colonization and that a cagZ mutant outcompeted the wild-type strain in a coinfection analysis. Our work supports the conclusion that RIVET is a valuable tool for identifying H. pylori factors with roles in host colonization.
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Abstract
The synthesis of "typical" hexa-acylated lipid A occurs via a nine-step enzymatic pathway, which is generally well conserved throughout all gram-negative bacteria. One exception to the rule is Helicobacter pylori, which has only eight homologs to the nine lipid A biosynthetic enzymes. The discrepancy occurs toward the end of the pathway, with H. pylori containing only a single putative secondary acyltransferase encoded by jhp0265. In Escherichia coli K-12, two late acyltransferases, termed LpxL and LpxM, are required for the biosynthesis of hexa-acylated lipid A. Detailed biochemical and genetic analyses reveal that H. pylori Jhp0265 (the protein encoded by jhp0265) is in fact an LpxL homolog, capable of transferring a stearoyl group to the hydroxyl group of the 2' linked fatty acyl chain of lipid A. Despite the lack of a homolog to LpxM in the H. pylori genome, the organism synthesizes a hexa-acylated lipid A species, suggesting that an equivalent enzyme exists. Using radiolabeled lipid A substrates and acyl-acyl carrier protein as the fatty acyl donor, we were able to confirm the presence of a second H. pylori late acyl transferase by biochemical assays. After synthesis of the hexa-acylated lipid A species, several modification enzymes then function to produce the major lipid A species of H. pylori that is tetra-acylated. Jhp0634 was identified as an outer membrane deacylase that removes the 3'-linked acyl chains of H. pylori lipid A. Together, this work elucidates the biochemical machinery required for the acylation and deacylation of the lipid A domain of H. pylori lipopolysaccharide.
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33
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Amundsen SK, Fero J, Hansen LM, Cromie GA, Solnick JV, Smith GR, Salama NR. Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization. Mol Microbiol 2008; 69:994-1007. [PMID: 18573180 PMCID: PMC2680919 DOI: 10.1111/j.1365-2958.2008.06336.x] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.
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Affiliation(s)
- Susan K. Amundsen
- Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle WA, 98109
| | - Jutta Fero
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle WA, 98109
| | - Lori M. Hansen
- Departments of Internal Medicine and Medical Microbiology & Immunology, and Center for Comparative Medicine, University of California, Davis CA, 95616, USA
| | - Gareth A. Cromie
- Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle WA, 98109
| | - Jay V. Solnick
- Departments of Internal Medicine and Medical Microbiology & Immunology, and Center for Comparative Medicine, University of California, Davis CA, 95616, USA
| | - Gerald R. Smith
- Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle WA, 98109
| | - Nina R. Salama
- Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle WA, 98109
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34
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Castillo AR, Arevalo SS, Woodruff AJ, Ottemann KM. Experimental analysis of Helicobacter pylori transcriptional terminators suggests this microbe uses both intrinsic and factor-dependent termination. Mol Microbiol 2008; 67:155-70. [PMID: 18078442 DOI: 10.1111/j.1365-2958.2007.06033.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
In this study, we report experimental analysis of transcriptional terminators in the human pathogen Helicobacter pylori. Previous bioinformatics approaches came to differing conclusions regarding transcriptional termination in this bacterium. We used a reporter construct, the tnpR-encoded resolvase, to assess terminators. In our first experiments, we found that a subset of previously predicted intrinsic terminators for H. pylori are functional. In our second experiments, we used an unbiased screen to identify putative terminators and then characterized 18 of these. We found that these putative terminators overlap genomic regions that are either intergenic or intragenic. Using reverse transcription PCR, we showed that an intergenic terminator and an intragenic antisense terminator function at their endogenous loci. Additionally, we found that putative terminators contain features of both intrinsic and Rho-dependent termination, but that intrinsic terminators define the majority. We were unable to delete rho, however, in H. pylori, suggesting that it is essential and likely important. Finally, we carried out a mutational analysis of one of our randomly identified terminators that has both intrinsic and Rho-dependent features, and found that they are both functional. In conclusion, we found that H. pylori possesses numerous Rho-dependent and intrinsic terminators including some found in intragenic regions.
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Affiliation(s)
- Andrea R Castillo
- Department of Environmental Toxicology, University of California, Santa Cruz, CA, USA.
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35
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Cornally D, Mee B, MacDonaill C, Tipton KF, Kelleher D, Windle HJ, Henehan GTM. Aldo-keto reductase from Helicobacter pylori--role in adaptation to growth at acid pH. FEBS J 2008; 275:3041-50. [PMID: 18445038 DOI: 10.1111/j.1742-4658.2008.06456.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Pyridine-linked oxidoreductase enzymes of Helicobacter pylori have been implicated in the pathogenesis of gastric disease. Previous studies in this laboratory examined a cinnamyl alcohol dehydrogenase that was capable of detoxifying a range of aromatic aldehydes. In the present work, we have extended these studies to identify and characterize an aldoketo reductase (AKR) enzyme present in H. pylori. The gene encoding this AKR was identified in the sequenced strain of H. pylori, 26695. The gene, referred to as HpAKR, was cloned and expressed in Escherichia coli as a His-tag fusion protein, and purified using nickel chelate chromatography. The gene product (HpAKR) has been assigned to the AKR13C1 family, although it differs in specificity from the two other known members of this family. The enzyme is a monomer with a molecular mass of approximately 39 kDa on SDS/PAGE. It reduces a range of aromatic aldehyde substrates with high catalytic efficiency, and exhibits dual cofactor specificity for both NADPH and NADH. HpAKR can function over a broad pH range (pH 4-9), and has a pH optimum of 5.5. It is inhibited by sodium valproate. Its substrate specificity complements that of the cinnamyl alcohol dehydrogenase activity in H. pylori, giving the organism the capacity to reduce a wide range of aldehydes. Generation of an HpAKR isogenic mutant of H. pylori demonstrated that HpAKR is required for growth under acidic conditions, suggesting an important role for this enzyme in adaptation to growth in the gastric mucosa. This AKR is a member of a hitherto little-studied class.
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Affiliation(s)
- Denise Cornally
- School of Food Science and Environmental Health, Dublin Institute of Technology, Ireland
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36
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Baltrus DA, Guillemin K, Phillips PC. Natural transformation increases the rate of adaptation in the human pathogen Helicobacter pylori. Evolution 2007; 62:39-49. [PMID: 17976191 DOI: 10.1111/j.1558-5646.2007.00271.x] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Gene exchange between individuals can lead to profound evolutionary effects at both the genomic and population levels. These effects have sparked widespread interest in examining the specific adaptive benefits of recombination. Although this work has primarily focused on the benefits of sex in eukaryotes, it is assumed that similar benefits of genetic exchange apply across eukaryotes and prokaryotes. Here we report a direct test of this assumption using the naturally transformable human gastric pathogen Helicobacter pylori as a model organism. We show that genetic exchange accelerates adaptation to a novel laboratory environment within bacterial populations and that a general adaptive advantage exists for naturally transformable strains when transfer occurs among conspecific backgrounds. This finding demonstrates that there are generalized benefits to adaptation in both eukaryotes and prokaryotes even though the underlying processes are mechanistically different.
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Affiliation(s)
- David A Baltrus
- Center for Ecology and Evolution, University of Oregon, Eugene, OR 97403, USA
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37
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Rader BA, Campagna SR, Semmelhack MF, Bassler BL, Guillemin K. The quorum-sensing molecule autoinducer 2 regulates motility and flagellar morphogenesis in Helicobacter pylori. J Bacteriol 2007; 189:6109-17. [PMID: 17586631 PMCID: PMC1951907 DOI: 10.1128/jb.00246-07] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
The genome of the gastric pathogen Helicobacter pylori contains a homologue of the gene luxS, which has been shown to be responsible for production of the quorum-sensing signal autoinducer 2 (AI-2). We report here that deletion of the luxS gene in strain G27 resulted in decreased motility on soft agar plates, a defect that was complemented by a wild-type copy of the luxS gene and by the addition of cell-free supernatant containing AI-2. The flagella of the luxS mutant appeared normal; however, in genetic backgrounds lacking any of three flagellar regulators--the two-component sensor kinase flgS, the sigma factor sigma28 (also called fliA), and the anti-sigma factor flgM--loss of luxS altered flagellar morphology. In all cases, the double mutant phenotypes were restored to the luxS+ phenotype by the addition of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD), which cyclizes to form AI-2. Furthermore, in all mutant backgrounds loss of luxS caused a decrease in transcript levels of the flagellar regulator flhA. Addition of DPD to luxS cells induced flhA transcription in a dose-dependent manner. Deletion of flhA in a wild-type or luxS mutant background resulted in identical loss of motility, flagella, and flagellar gene expression. These data demonstrate that AI-2 functions as a secreted signaling molecule upstream of FlhA and plays a critical role in global regulation of flagellar gene transcription in H. pylori.
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Affiliation(s)
- Bethany A Rader
- Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
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Levine SM, Lin EA, Emara W, Kang J, DiBenedetto M, Ando T, Falush D, Blaser MJ. Plastic cells and populations: DNA substrate characteristics in Helicobacter pylori transformation define a flexible but conservative system for genomic variation. FASEB J 2007; 21:3458-67. [PMID: 17567566 DOI: 10.1096/fj.07-8501com] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Helicobacter pylori, bacteria that colonize the human gastric mucosa, are naturally competent for transformation by exogenous DNA, and show a panmictic population structure. To understand the mechanisms involved in its horizontal gene transfer, we sought to define the interval required from exposure to substrate DNA until DNA uptake and expression of a selectable phenotype, as well as the relationship of transforming fragment length, concentration, homology, symmetry, and strandedness, to the transformation frequency. We provide evidence that natural transformation in H. pylori differs in efficiency among wild-type strains but is saturable and varies with substrate DNA length, symmetry, strandedness, and species origin. We show that H. pylori cells can be transformed within one minute of contact with DNA, by DNA fragments as small as 50 bp, and as few as 5 bp on one flank of a selectable single nucleotide mutation is sufficient substrate for recombination of a transforming fragment, and that double-stranded DNA is the preferred (1000-fold >single-stranded) substrate. The high efficiency of double-stranded DNA as transformation substrate, in conjunction with strain-specific restriction endonucleases suggests a model of short-fragment recombination favoring closest relatives, consistent with the observed H. pylori population biology.
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Affiliation(s)
- Steven M Levine
- Dept. of Medicine, New York University School of Medicine, New York, NY 10016, USA
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Snelling WJ, Moran AP, Ryan KA, Scully P, McGourty K, Cooney JC, Annuk H, O'Toole PW. HorB (HP0127) is a gastric epithelial cell adhesin. Helicobacter 2007; 12:200-9. [PMID: 17492999 DOI: 10.1111/j.1523-5378.2007.00499.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND The Helicobacter pylori protein HorB (encoded by HP0127) is a member of a paralogous family that includes the adhesins BabA, AlpA, AlpB, and HopZ, which contribute to adhesion to gastric epithelial cells. Of the verified H. pylori porins, the HorB sequence is most similar to that of HopE, but the function of HorB is unknown. The aim of our study was to investigate the role of HorB in H. pylori gastric epithelial cell adhesion. MATERIALS AND METHODS We disrupted the horB gene in H. pylori and measured the adhesion to gastric epithelial cells (AGS cells). We then assessed the effect that HorB disruption had on lipopolysaccharide (LPS) O-chain production and Lewis x and Lewis y antigen expression. A HorB mutant in the mouse-adapted strain H. pylori SS1 was created by marker exchange and mouse stomach colonization was quantified. Using reverse transcription polymerase chain reaction, human gastric biopsy material from H. pylori-infected patients was then examined for expression of the horB gene. RESULTS Disruption of the horB gene reduced H. pylori adhesion by more than twofold. Adhesion in the horB knockout strain was restored to wild-type levels by re-introduction of HorB into the chromosome. Disruption of HorB reduced production of LPS O-chains and lowered the level of expression of Lewis x and Lewis y antigens. Insertional mutagenesis of the horB gene in H. pylori SS1 reduced mouse stomach colonization threefold. Finally, expression of the horB gene was detected in human gastric biopsy material from H. pylori-infected patients. CONCLUSIONS From these data we conclude that HorB has a role in H. pylori adhesion during infection.
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Affiliation(s)
- William J Snelling
- Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork, Ireland.
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Fadel S, Eley A. Chlamydia trachomatis OmcB protein is a surface-exposed glycosaminoglycan-dependent adhesin. J Med Microbiol 2007; 56:15-22. [PMID: 17172511 DOI: 10.1099/jmm.0.46801-0] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The OmcB protein of Chlamydia trachomatis is a cysteine-rich outer membrane polypeptide with important functional, structural and antigenic properties. The entire gene encoding the OmcB protein from C. trachomatis serovar LGV1 was cloned and expressed in Escherichia coli and the full-length protein used to raise polyclonal antibodies. Recombinant OmcB was used to show that OmcB is a surface-exposed protein that functions as a chlamydial adhesin. Infectivity inhibition assays carried out using HeLa cells with serovar LGV1 in the presence of purified anti-OmcB serum showed inhibition of infectivity, suggesting that some of the OmcB was surface exposed. Moreover, using recombinant OmcB in infectivity inhibition assays resulted in 70% inhibition of infectivity, confirming that OmcB plays a role as an adhesin in C. trachomatis. Furthermore, recombinant OmcB protein bound to the surface of HeLa and Hec1B cells, but binding to glycosaminoglycan (GAG)-deficient cells (pgsA-745 and pgsD-677) was markedly reduced, indicating that OmcB binds to GAG-like receptors on host cells.
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Affiliation(s)
- Sanaa Fadel
- Henry Wellcome Laboratories for Medical Research, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK
| | - Adrian Eley
- Henry Wellcome Laboratories for Medical Research, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK
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Langford ML, Zabaleta J, Ochoa AC, Testerman TL, McGee DJ. In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site. Helicobacter 2006; 11:477-93. [PMID: 16961811 PMCID: PMC2963585 DOI: 10.1111/j.1523-5378.2006.00441.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
BACKGROUND Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS This complementation strategy should greatly facilitate genetic experiments in H. pylori.
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Affiliation(s)
| | - Jovanny Zabaleta
- Department of Pathology and Tumor Immunology Program, Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, New Orleans, LA 70112, USA
| | - Augusto C. Ochoa
- Department of Pathology and Tumor Immunology Program, Stanley S. Scott Cancer Center, Louisiana State University, Health Sciences Center, New Orleans, LA 70112, USA
- Department of Pediatrics, Louisiana State University, Health Sciences Center, New Orleans, LA 70112, USA
| | - Traci L. Testerman
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
| | - David J. McGee
- Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
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42
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Mouery K, Rader BA, Gaynor EC, Guillemin K. The stringent response is required for Helicobacter pylori survival of stationary phase, exposure to acid, and aerobic shock. J Bacteriol 2006; 188:5494-500. [PMID: 16855239 PMCID: PMC1540029 DOI: 10.1128/jb.00366-06] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The gastric pathogen Helicobacter pylori must adapt to fluctuating conditions in the harsh environment of the human stomach with the use of a minimal number of transcriptional regulators. We investigated whether H. pylori utilizes the stringent response, involving signaling through the alarmone (p)ppGpp, as a survival strategy during environmental stresses. We show that the H. pylori homologue of the bifunctional (p)ppGpp synthetase and hydrolase SpoT is responsible for all cellular (p)ppGpp production in response to starvation conditions. Furthermore, the H. pylori spoT gene complements the growth defect of Escherichia coli mutants lacking (p)ppGpp. An H. pylori spoT deletion mutant is impaired for stationary-phase survival and undergoes a premature transformation to a coccoid morphology. In addition, the spoT deletion mutant is unable to survive specific environmental stresses, including aerobic shock and acid exposure, which are likely to be encountered by this bacterium during infection and transmission.
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Affiliation(s)
- Kyle Mouery
- Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
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Baltrus DA, Guillemin K. Multiple phases of competence occur during the Helicobacter pylori growth cycle. FEMS Microbiol Lett 2006; 255:148-55. [PMID: 16436074 DOI: 10.1111/j.1574-6968.2005.00066.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The gastric pathogen Helicobacter pylori undergoes genetic exchange at unusually high frequencies, primarily through natural transformation. Despite progress toward understanding the molecular mechanism of natural transformation in H. pylori, little is known about how competence is regulated or its relationship to DNA release. By measuring transformation incrementally throughout the growth curve, we show that H. pylori exhibits a novel pattern of competence with distinct peaks of transformation during both logarithmic and stationary growth phases. Furthermore, different H. pylori strains vary in the presence and timing of their competence peaks. We also examined the process of DNA release in relation to competence. Although extensive DNA release does not occur until late stationary phase, sufficient genomic DNA was present during the logarithmic phase to yield measurable transformants. These results demonstrate that the state of competence in H. pylori occurs in an unprecedented pattern during the growth curve with no clear relationship to DNA release.
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Affiliation(s)
- David A Baltrus
- Center for Ecology and Evolution, University of Oregon, Eugene, USA
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44
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Tran AX, Whittimore JD, Wyrick PB, McGrath SC, Cotter RJ, Trent MS. The lipid A 1-phosphatase of Helicobacter pylori is required for resistance to the antimicrobial peptide polymyxin. J Bacteriol 2006; 188:4531-41. [PMID: 16740959 PMCID: PMC1482963 DOI: 10.1128/jb.00146-06] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.
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Affiliation(s)
- An X Tran
- Department of Microbiology, J.H. Quillen College of Medicine, Johnson City, TN 37614, USA
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45
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Bourzac KM, Satkamp LA, Guillemin K. The Helicobacter pylori cag pathogenicity island protein CagN is a bacterial membrane-associated protein that is processed at its C terminus. Infect Immun 2006; 74:2537-43. [PMID: 16622188 PMCID: PMC1459751 DOI: 10.1128/iai.74.5.2537-2543.2006] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Helicobacter pylori infects nearly half the world's population and is associated with a spectrum of gastric maladies. Infections with cytotoxin-associated gene pathogenicity island (cag PAI)-containing strains are associated with an increased risk for gastric cancer. The cag PAI contains genes encoding a type IV secretion system (T4SS) and a delivered effector, CagA, that becomes tyrosine phosphorylated upon delivery into host cells and initiates changes in cell signaling. Although some cag PAI genes have been shown to be required for CagA delivery, a subset of which are homologues of T4SS genes from Agrobacterium tumefaciens, the majority have no known function or homologues. We have performed a detailed investigation of one such cag PAI protein, CagN, which is encoded by the gene HP0538. Our results show that CagN is not delivered into host cells and instead is associated with the bacterial membrane. We demonstrate that CagN is cleaved at its C terminus by a mechanism that is independent of other cag PAI proteins. Finally, we show that a delta cagN mutant is not impaired in its ability to deliver CagA to gastric epithelial cells and initiate cell elongation.
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Affiliation(s)
- Kevin M Bourzac
- Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
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46
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Croxen MA, Sisson G, Melano R, Hoffman PS. The Helicobacter pylori chemotaxis receptor TlpB (HP0103) is required for pH taxis and for colonization of the gastric mucosa. J Bacteriol 2006; 188:2656-65. [PMID: 16547053 PMCID: PMC1428400 DOI: 10.1128/jb.188.7.2656-2665.2006] [Citation(s) in RCA: 133] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.
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Affiliation(s)
- Matthew A Croxen
- Department of Microbiology, Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada
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47
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Karnholz A, Hoefler C, Odenbreit S, Fischer W, Hofreuter D, Haas R. Functional and topological characterization of novel components of the comB DNA transformation competence system in Helicobacter pylori. J Bacteriol 2006; 188:882-93. [PMID: 16428391 PMCID: PMC1347336 DOI: 10.1128/jb.188.3.882-893.2006] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Helicobacter pylori is one of the most diverse bacterial species known. A rational basis for this genetic variation may be provided by its natural competence for genetic transformation and high-frequency recombination. Many bacterial competence systems have homology with proteins that are involved in the assembly of type IV pili and type II secretion systems. In H. pylori, DNA uptake relies on a transport system related to type IV secretion systems (T4SS) designated the comB system. The prototype of a T4SS in Agrobacterium tumefaciens consists of 11 VirB proteins and VirD4, which form the core unit necessary for the delivery of single proteins or large nucleoprotein complexes into target cells. In the past we identified proteins ComB4 and ComB7 through ComB10 as being involved in the process of DNA uptake in H. pylori. In this study we identified and functionally characterized further (T4SS-homologous) components of the comB transformation competence system. By combining computer prediction modeling, experimental topology determination, generation of knockout strains, and genetic complementation studies we identified ComB2, ComB3, and ComB6 as essential components of the transformation apparatus, structurally and functionally homologous to VirB2, VirB3, and VirB6, respectively. comB2, comB3, and comB4 are organized as a separate operon. Thus, for the H. pylori comB system, all T4SS core components have been identified except for homologues to VirB1, VirD4, VirB5, and VirB11.
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Affiliation(s)
- Arno Karnholz
- Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie, Ludwig-Maximilians-Universität, Pettenkoferstr. 9a, D-80336 München, Germany
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Abstract
BACKGROUND Most strategies for direct mutagenesis of Helicobacter pylori primarily involve genomic DNA cloning which is a time-consuming and expensive technique. METHODS To make a gene replacement, we propose a strategy using polymerase chain reaction (PCR) amplicons to allow a double homologous recombination in the genome of H. pylori. Different strains were used to validate this strategy and we describe how the amplicon insertion was made with accuracy. Moreover, we looked for the shortest homologous sequence needed to allow a specific gene replacement in H. pylori without any deletion, insertion or mutation at the recombination site. All of the experiments were performed at the flaA locus, whose gene encodes the major flagellin. RESULTS Amplicons bearing 500 or 150 bp flanking regions of flaA on each side (depending on the strain) were sufficient to allow the specific insertion of a 1173 bp chloramphenicol cassette into the genome of H. pylori. The insertion was accurate with no substitutions at the insertion locus. CONCLUSIONS This information opens the door to other strategies for mutagenesis used for the identification of virulence factors without deleting genes, which would not be based on a negative screening system. For example, they could be useful in performing protein fusion for a better understanding of the virulence factor's mechanism.
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Affiliation(s)
- Slovénie Pyndiah
- INSERM ESPRI 2004, Laboratoire de Bactériologie, UniversitéVictor Segalen Bordeaux 2, 33076 Bordeaux cedex, France
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49
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The 2005 Annual Meeting of the British Society of Gastroenterology. Birmingham, United Kingdom, 14-17 March 2005. Abstracts. Gut 2005; 54 Suppl 2:A1-A117. [PMID: 15764805 PMCID: PMC1867799] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/08/2022]
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50
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McGee DJ, Langford ML, Watson EL, Carter JE, Chen YT, Ottemann KM. Colonization and inflammation deficiencies in Mongolian gerbils infected by Helicobacter pylori chemotaxis mutants. Infect Immun 2005; 73:1820-7. [PMID: 15731083 PMCID: PMC1064941 DOI: 10.1128/iai.73.3.1820-1827.2005] [Citation(s) in RCA: 80] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.
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Affiliation(s)
- David J McGee
- Department of Microbiology & Immunology, University of South Alabama College of Medicine, 307 N. University Blvd., Mobile, AL 36688, USA.
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